Work will continue on the mode of action of peptide A1 of cholera toxin which activates adenylyl cyclase in broken cell systems providing that NAD and ATP are present and that a certain soluble cytoplasmic factor, apparently a protein, is present. Most work will involve lysed pigeon erythrocytes as a source both of adenyly cyclase and of the cytoplasmic factor. Our major thrust will be to purify and characterize the cytoplasmic factor using standard protein fractionation procedures. A second goal is to purify cholera toxin with subunit A intact and to study its effect upon intact cells. We are particularly interested to discover whether nicking is required for toxicity, a question relevant to the mode of entry of A1 into the cytoplasm and reminiscent of the parallel problem concerning diphtheria toxin. To find conditions under which intact toxin accumulates, we propose to grow C. vibrio in the presence of proteolytic inhibitors and to employ protease mutants of the bacterium. BIBLIOGRAPHIC REFERENCES: D.M. Gill. The arrangement of subunits in cholera toxin. Biochemistry 15, 1242-1248. Appendix: gel scans (76). D.M. Gill. Protein toxins that act within cells. Bull. Inst. Pasteur. 74. 65-84. (76).